Protein kinases mediate intracellular signal transduction by causing a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor involved in a signaling pathway. There are a number of kinases and pathways through which extracellular and other stimuli cause a variety of cellular responses to occur inside the cell. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α)), growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis and regulation of cell cycle.
Many disease states are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include autoimmune diseases, inflammatory diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease and hormone-related diseases. Thus, an understanding of the structure, function, and inhibition of kinase activity could lead to useful human therapeutics.
Among medically important kinases are the serine/threonine kinases. The serine/threonine kinase family include the mammalian mitogen-activated protein (MAP) kinases. MAP kinases are activated by dual phosphorylation of threonine and tyrosine at the Thr-X-Tyr segment in the activation loop. Members of the MAP kinase family also share sequence similarity and conserved structural domains, and include the extracellular-signal regulated kinases (ERKs), Jun N-terminal kinases (JNKs) and p38 kinases. MAP kinases also phosphorylate various substrates including transcription factors, which in turn regulate the expression of specific sets of genes and mediate a specific response to the stimulus.
Another important group in the serine/threonine kinase family includes a subgroup of three closely related serine/threonine protein kinases, the Aurora kinases. The Aurora kinases play a key role in protein phosphorylation events that regulate the mitotic phase of the cell cycle. Aurora-2, for example, is up-regulated during the M phase of the cell cycle and localizes to the spindle pole during mitosis, suggesting a possible involvement in centrosomal functions. The Aurora kinases share a common structure, including a highly-conserved catalytic domain, and a very short N-terminal domain that varies in size (R. Giet and C. Prigent, J. Cell Sci., 112, pp. 3591-'3601 (1999)). The N-terminal domains do not share any sequence similarity. The Aurora kinases are overexpressed in various types of cancer, such as colon, breast and other solid tumors (for a review see T. M. Goepfert and B. R. Brinkley, Curr. Top. Dev. Biol., 49, pp. 331-342 (2000)). Even more importantly, both the Aurora-1 and -2 genes are amplified in breast and colorectal cancers whereas the Aurora-3 gene is located in a region that is rearranged or deleted in several cancer cells. Overexpression of Aurora-2 in rodent fibroblasts induces transformation, indicating that Aurora-2 is oncogenic. Recently, Aurora-2 mRNA expression has been linked to chromosomal instability in human breast cancers (Y. Miyoshi et al., Int. J. Cancer, 92, pp. 370-373 (2001)).
Accordingly, there has been an interest in finding inhibitors of Aurora-1, Aurora-2 or Aurora-3 that are effective as therapeutic agents. A challenge has been to find protein kinase inhibitors that act in a selective manner for the Aurora family kinases. Since there are numerous protein kinases involved in a variety of cellular responses, non-selective inhibitors may lead to undesirable side effects. In this regard, the three-dimensional structure of the kinase would assist in the rational design of inhibitors. The determination of the amino acid residues in Aurora-2 binding pockets and the determination of the shape of those binding pockets would allow one to design selective inhibitors that bind favorably to this class of enzymes. The determination of the amino acid residues in Aurora-2 binding pockets and the determination of the shape of those binding pockets would also allow one to design inhibitors that can bind selectively to Aurora-1, Aurora-2 or Aurora-3, or any combination thereof.
Despite the fact that the genes for various Aurora-1, Aurora-2 and Aurora-3 have been isolated and the amino acid sequences of Aurora-1, Aurora-2 and Aurora-3 proteins are known, the X-ray crystal structural coordinate information of Aurora-1, Aurora-2 or Aurora-3 protein has not yet been described. Such information would be useful in identifying and designing therapeutic inhibitors of the Aurora kinases or homologues thereof.